Bilayers

ABSTRACT

A method for producing a bilayer of amphipathic molecules comprising providing a hydrated support and providing a hydrophilic body, and bringing the hydrated support and hydrophilic body into contact to form a bilayer of amphipathic molecules. A bilayer produced by the method of the invention, and uses of the bilayer.

The present invention relates to a bilayer, such as a lipid bilayer, toa method of producing a bilayer, to the use of a bilayer and toapparatus to produce and/or use a bilayer.

Artificial planar lipid bilayers serve as simplified models ofbiological membranes and are widely used for the electricalcharacterisation of ion-channels and protein pores. Ion-channels are adiverse group of membrane proteins that selectively control the movementof specific ions across cell membranes, establishing voltage andelectrochemical gradients that are fundamental to a wide variety ofbiological processes. In humans, ion-channels regulate everything fromheartbeat and muscle contraction to hormone secretion and the release ofneurotransmitters. Defective ion channel function is implicated in agrowing list of disorders, including cardiac arrhythmia, periodicparalysis, epilepsy and diabetes (Ashcroft, F. M. 2000, Academic Press,San Diego; Ashcroft, F. M. 2006, Nature 440, 440-447; Kass, R. S. 2005,Journal of Clinical Investigation 115, 1986-1989). Protein pores arenon-specific channels that allow molecules to pass across cellmembranes. Protein pores can be exploited for many applications such asmolecular sensing (Bayley, H. et al., 2000, Advanced Materials 12,139-142; Bayley, H. & Cremer, P. S. 2001. Nature 413, 226-230) and DNAsequencing (Kasianowicz, J. J. et al., 1996. Proceedings of the NationalAcademy of Sciences of the United States of America 93, 13770-13773;Howorka, S. et al., 2001. Nature Biotechnology 19, 636-639; Astier, Y.2006. Journal of the American Chemical Society 128, 1705-1710).

Single-channel recording (SCR) of individual proteins is a powerfulmeans of studying channel protein function (Sakmann, B. & Neher, E.1995. Plenum Press, New York; London). Single-channel recording measureschanges in ion-current through single protein channels, and can examinevoltage dependence, gating behaviour, ligand binding affinity, and ionselectivity at the single-molecule level. Consequently, single-channelrecording can help determine the molecular basis of an ion-channeldisease. It is also an important technique for the development of newdrugs specifically targeting channelopathies, and for screening othermedicines for unwanted side-effects (Ashcroft, F. M. 2006. Nature 440,440-447; Roden, D. M. 2004. New England Journal of Medicine 350,1013-1022). Advances in these areas require much higher throughputassays of ion-channel behaviour than are currently available.

Single-channel recording typically uses either patch-clamping (Sakmann,B. & Neher, E. 1984. Annual Review of Physiology 46, 455-472) orartificial planar lipid bilayers (Mueller, P. et al., 1962. Nature 194,979-980; White, S. H. 1986. ed. Miller, C. Plenum Press: New York).Although other methods may also be used, including excised-patch,tip-dip and on-chip methods.

Patch-clamping of whole cells is a versatile and sensitive means ofexamining channels, but is time-consuming and often complicated by theheterogeneous nature of cell membranes. In contrast, artificial planarlipid bilayers control the constituents of the system and can be used tostudy purified proteins. Planar lipid bilayers are usually formed eitherby painting, where a solution of lipid in an organic solvent is directlyapplied to an aperture separating two aqueous compartments (Mueller, P.et al., 1962. Nature 194, 979-980; White, S. H. 1986. ed. Miller, C.Plenum Press: New York), or variants of the Langmuir-Blodgett technique,where two air/water monolayers are raised past an aperture (Montal, M. &Mueller, P. 1972. Proceedings of the National Academy of Sciences of theUnited States of America 69, 3561-3566). Although widely used, planarlipid bilayers are difficult to prepare, and their short lifetimeprohibits their use in many situations.

Alternative emulsion-based approaches to forming bilayers have also beenproposed (Tsofina, L. M. et al., 1966. Nature 212, 681-683), wherebilayers are created between aqueous surfaces immersed in a solution oflipid in oil. When immersed in an immiscible lipid/oil solution, aqueoussurfaces spontaneously self-assemble a lipid monolayer (Cevc, G. 1993.Phospholipids handbook, ed. Cevc, G., Marcel Dekker, New York); Seddon,J. M. & Templer, R. H. 1995. eds. Lipowsky, R. & Sackmann, E., Elsevier,Amsterdam, Oxford), and when monolayers from two aqueous components arebrought into contact they can ‘zip’ together to form a lipid bilayer(Tien, H. T. 1974. M. Dekker, New York; Fujiwara, H. et al., 2003.Journal of Chemical Physics 119, 6768-6775). Recent studies have shownthat microfluidic flows (Malmstadt, N. et al., 2006. Nano Letters 6,1961-1965; Funakoshi, K. et al., 2006. Analytical Chemistry 78,8169-8174) and droplets (Funakoshi, K. et al., 2006. AnalyticalChemistry 78, 8169-8174; Holden, M. A. et al., 2007. Journal of theAmerican Chemical Society p8650-5) can be contacted in a lipid/oilsolution to create bilayers suitable for single-channel recordingexperiments.

According to a first aspect of the invention there is provided a methodfor producing a bilayer of amphipathic molecules comprising the stepsof:

-   -   (i) providing a hydrated support in a hydrophobic medium,        wherein the hydrophobic medium contains amphipathic molecules        and a first monolayer of amphipathic molecules is present on the        surface of the hydrated support;    -   (ii) providing a hydrophilic body in a hydrophobic medium,        wherein the hydrophobic medium contains amphipathic molecules        and a second monolayer of amphipathic molecules is present on        the surface of the hydrophilic body; and    -   (iii) bringing the first monolayer and the second monolayer into        contact to form a bilayer of amphipathic molecules.

Preferably step (i) of the first method of the invention comprisesproviding a hydrated support in a hydrophobic medium, wherein thehydrophobic medium contains amphipathic molecules, and then forming afirst monolayer of amphipathic molecules on the surface of the hydratedsupport.

Preferably step (ii) of the first method of the invention comprisesproviding a hydrophilic body in a hydrophobic medium, wherein thehydrophobic medium contains amphipathic molecules, and then forming asecond monolayer of amphipathic molecules on the surface of thehydrophilic body.

According to a second aspect of the invention there is provided a methodfor producing a bilayer of amphipathic molecules comprising the stepsof:

-   -   (i) providing a hydrated support containing amphipathic        molecules in a hydrophobic medium, wherein a first monolayer of        amphipathic molecules is present on the surface of the hydrated        support;    -   (ii) providing a hydrophilic body containing amphipathic        molecules in a hydrophobic medium, wherein a second monolayer of        amphipathic molecules is present on the surface of the        hydrophilic body; and    -   (iii) bringing the first monolayer and the second monolayer into        contact to form a bilayer of amphipathic molecules.

Preferably step (i) of the second method of the invention comprisesproviding a hydrated support containing amphipathic molecules in ahydrophobic medium, and then forming a first monolayer of amphipathicmolecules on the surface of the hydrated support.

Preferably step (ii) of the second method of the invention comprisesproviding a hydrophilic, body containing amphipathic molecules in ahydrophobic medium, and then forming a second monolayer of amphipathicmolecules on the surface of the hydrophilic body.

It is found that the method of both the first and the second aspect ofthe invention spontaneously forms a bilayer of amphipathic moleculeswhich has the added advantage that it is stable over long periods oftime and when subjected to environmental and/or physical stress.

The life-time of the bilayer of amphipathic molecules made according toany method of the invention may be greater than about 1 hour, 5 hours,10 hours, 24 hours, 2 days, 1 week, 1 month, 2 months, 3 months or more.

Preferably the method of the invention forms bilayers with at leastabout 90% efficiency, more preferably with about 95%, 98% or 99%efficiency.

Preferably the bilayers form within 1 minute of contact between themonolayer on the hydrated support and the monolayer on hydrophilic body.

The bilayer of amphipathic molecules may be capable of withstandingphysical shock, for example, the bilayer may be stable after beingdropped one or more times from heights greater than 0.5 metres or 1metre. This bilayer stability makes bilayers produced by any method ofthe invention easier to work with than bilayers produced by knownconventional methods.

The surprisingly long life-time and stability of the bilayer ofamphipathic molecules produced by any method of the invention has thebenefit that a user is able to set up the bilayer and use it in longterm and/or multiple experiments. It also has the benefit of beingcapable of being more readily used in a portable device, for exampleoutside of a controlled laboratory environment, where the physicalenvironment is less controllable or predictable, than conventionalbilayers.

Preferably a monolayer of amphipathic molecules self assembles on thehydrated support and the hydrophilic body when each is placed in ahydrophobic medium containing amphipathic molecules.

The orientation of the amphipathic molecules in the monolayers meansthat a bilayer forms when the monolayers are brought into contact.

Amphipathic molecules have both a hydrophilic group and a hydrophobicgroup. In the monolayers, formed in any method of the invention, theamphipathic molecules are aligned on the surface of the hydrophilic bodyand the hydrated substrate with the hydrophilic groups (or “heads”)towards the water interface and the hydrophobic groups (or “tails”) awayfrom the water interface.

The amphipathic molecules used in any method of the invention may belipid molecules, in particular, surfactant molecules may be used. Thelipid molecules may be selected from the group comprising fatty acyls,glycerolipids, glycerophospholipids, sphingolipids, sterol lipids,prenol lipids, saccharolipids, polyketides, phospholipids, glycolipidsand cholesterol.

The lipid may include any of the group comprising monoolein;1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC);1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC);1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE);1-palmitoyl-2-oleoyl-phosphatidylethanolamine; and1-palmitoyl-2-oleoylphosphatidylglycerol (POPE/POPG) mixtures; ormixtures thereof.

The amphipathic molecules in the monolayers or bilayers may be of thesame or different types. For example, each monolayer of the bilayer maycomprise a different type of amphipathic molecule such that the bilayerproduced is asymmetric. An asymmetric bilayer may be produced using themethod of the first aspect of the invention wherein the first monolayeris formed on the hydrated support in a first hydrophobic mediumcontaining a first type of amphipathic molecule. The second monolayer isformed on the hydrophilic body in a second hydrophobic medium containinga second type of amphipathic molecule. The first and second amphipathicmolecules may be different. The two monolayers, one on the hydrophilicbody and one on the hydrated support, are then brought together in athird hydrophobic medium which does not contain an amphipathic moleculeto form an asymmetric bilayer of amphipathic molecules. Alternatively,an asymmetric bilayer may be produced by the second method of theinvention by using a different type of amphipathic molecule in thehydrated support to that used in the hydrophilic body.

Alternatively, each monolayer may comprise the same type or mixtures oftypes of amphipathic molecules.

The hydrated support may comprise a solid or a semi-solid substrate. Theterms “solid” and “semi-solid” as used herein are understood to havetheir ordinary meaning to a person skilled in the art. Essentially theterm “solid” refers to a substrate that is rigid and resistant todeformation, and “semi-solid” refers to a substrate that has propertiesbetween those of a solid and a liquid. Preferably a semi-solid substratehas some degree of flexibility but is rigid enough to maintain its shapewhen placed in a container, and will not immediately conform to theshape of the container. An example of a semi-solid substrate is a gel.

Preferably the hydrated support is hydrophilic. Preferably a monolayerof amphipathic molecules will self assemble on the surface of thehydrated support if it is placed in the presence of amphipathicmolecules in a hydrophobic medium.

Alternatively, the hydrated support may not be hydrophilic and the lipidmonolayer may be formed by the attachment of lipid molecules to thesurface, for example, the surface of the support may be such thatmodified lipids will react with the surface and attach to form amonolayer.

The hydrated support may be porous or non-porous. Preferably thehydrated support is porous.

The hydrated support may be a hydrogel. The hydrogel may bephotocrosslinked.

The hydrated support may comprise agarose, polyacrylamide,[cross-linked] polyethylene glycol, nitro-cellulose, polycarbonate,anodisc material, polyethersulphone, cellulose acetate, nylon, Naphionmaterials, mesoporous silica, water and/or glass.

The hydrated support may be a protein or analyte separation gel, forexample, an electrophoresis gel. The separation gel may containproteins, DNA or other samples separated, for example, on the basis oftheir size, molecular weight or ionic properties.

The surface of the hydrated support which carries the amphipathicmolecule monolayer may be any suitable configuration. The surface may besubstantially flat, or the surface may be substantially uneven. Thesurface may be curved. The surface may be patterned.

The hydrated support may be partially or substantially transparent.Alternatively, the hydrated support may be largely opaque.

The hydrated support may comprise a substrate of any thickness,preferably from about 1 nm to about 10 cm, more preferably from about 1μm to about 1 cm, most preferably from about 100 μm to about 1 cm.

The hydrophilic body may be a liquid, solid, or semi-solid or a mixturethereof. Preferably the hydrophilic body comprises a droplet of aqueoussolution, such as water.

Where the hydrophilic body is a droplet of an aqueous solution itpreferably has a diameter of from about 5 nm to 10 cm or more,preferably from 1 μm to 1 mm. In one embodiment, droplets are around 100μm in diameter.

The hydrophilic body may comprise a hydrated solid or semi-solidsupport/substrate. The hydrophilic body may comprise a hydrogel, such ashydrated agarose.

The composition of the hydrophilic body is preferably controlled tocontain the correct salts to allow an electrical current to be carried,for example, NaCl, KCl, MgCl₂ and/or other salts may be included.

The hydrophilic body may also comprise common buffering agents tocontrol pH, for example, Bis-tris, Tris, Hepes, sodium phosphate and/orpotassium phosphate.

Salts may also be included for other reasons, for example, to stabiliseproteins, to control binding components, to control the osmotic gradientacross the bilayer and/or to activate fluorescent probes.

The hydrophilic body may also contain varying amounts of othercomponents, such as, sucrose or PEG which may be used to stabiliseosmotic stresses, fluorescent probes, microspheres or beads. Thehydrophilic bodies may also comprise denaturants such as urea orguanidine HCl.

Where more than one hydrophilic body is used each may be of the same ora different composition.

The hydrophobic medium may be an oil. The oil may be a hydrocarbon,which may be branched or unbranched, and may be substituted orunsubstituted. For example, the hydrocarbon may have from 5 to 20 carbonatoms, more preferably from 10 to 17 carbon atoms. Suitable oils includealkanes or alkenes, such as hexadecane, decane, pentane or squalene, orfluorinated oils, or silicone based oils, or carbon tetrachloride.

Preferably, the method of the invention uses a lipid (amphipathicmolecules) in oil (hydrophobic medium) solution. Preferably the lipid inoil solution contains from about 1 mg/ml to about 30 mg/ml of lipid inthe oil. Preferably, the lipid in oil solution contains about 5 mg/ml oflipid. Preferably the lipid/oil solution comprises1,2-diphytanoyl-sn-glycero-3-phophocholine (DPhPC) in n-hexadecane(C₁₆).

The terms “contacting” or “contact” used herein with reference to thecontacting of monolayers to form a bilayer are understood to mean actualphysical contact, and/or close enough proximity, to allow the assemblyof an amphipathic molecule bilayer from separate amphipathic moleculemonolayers. Preferably the contacting process is a form of Gibbs-plateauborder action.

The bilayer of amphipathic molecules may be from less than about 1 μm togreater than about 1 cm in diameter, preferably from about 5 μM to about5000 μm in diameter, more preferably from about 30 μm to about 3000 μmin diameter. The bilayer may be from about 5 μm to about 500 μm indiameter, more preferably from about 30 μm to about 300 μm in diameter.The skilled man will appreciate that the bilayer does not need to becircular. Where a non-circular bilayer is formed preferably the bilayerincludes a portion which has the aforementioned preferred diameter. Forexample, a non-circular bilayer according to the invention may include aportion which has diameter from about 1 μm to about 1 cm or more.Preferably, a non-circular bilayer according to the invention has onedimension which is from about 1 μm to about 1 cm or more.

The area of the bilayer of amphipathic molecules may be adjustable.Preferably the area of the bilayer layer is adjustable before, during,or in intervals between uses of the bilayer. The area of the bilayer maybe adjustable by increasing or decreasing the contact area between thehydrophilic body and the hydrated support. The contact area between thehydrophilic body and the hydrated support may be adjusted by moving thecentre of the hydrophilic body towards or away from the hydratedsupport, or by moving the hydrated support towards or away from thecentre of the hydrophilic body. Preferably to increase the area of thebilayer, the centre of the hydrophilic body is moved towards thehydrated support. Preferably to decrease the area of the bilayer, thecentre of the hydrophilic body is moved away from the hydrated support.Where an electrode is used to hold the hydrophilic body, the area of thebilayer may be increased or decreased by moving the electrode towards oraway from the hydrated support respectively. In an alternativeembodiment the position of the hydrophilic body may be controlled byusing an applied electric or magnetic field, by using light beams (suchas optical traps) or pressure. The area of the bilayer may be adjustedby increasing or decreasing the volume, size and/or shape of thehydrophilic body. Preferably the area of the bilayer can be changedwithout breaking the bilayer.

Preferably the area of the bilayer can be moved to any diameter between5 μm and about 500 μm, or an up to 100 fold change in bilayer size canbe achieved, in less than about 10 seconds, less than about 5 seconds,less than about 3 second, less than about 2 seconds, less than about 1second or less than about 0.5 seconds.

Controlling the area of the bilayer is surprisingly easy and fast.Controlling the area of the bilayer has the advantage of being able tocontrol the number/amount of membrane proteins that associate with thebilayer. Surprisingly, altering the area of the bilayer does notdenature or remove membrane-associated proteins from the bilayer.Indeed, if trans-membrane proteins are already inserted into the bilayerthey will become concentrated if the area of the bilayer is decreased.If contact between the hydrophilic body and the hydrated support isremoved, the bilayer will disassemble and any trans-membrane proteinlocated therein will be no longer present in the bilayer or eithermonolayer.

The method may include a stabilisation period to allow the monolayerand/or bilayer of amphipathic molecules to form. The stabilisationperiod may be to allow the system to reach equilibrium. Thestabilisation period may be from about 0 seconds to about 5 hours,preferably the stabilisation period is from about 10 seconds to about 1hour. Preferably the stabilisation period is about 15 minutes. In someembodiments if one component of the method, for example either the firstor second monolayer, has been left to stabilise after formation, thenthere may be no need to allow the other monolayer to stabilise beforeformation of the bilayer.

The stabilisation period has the advantage of reducing the chances ofthe hydrophilic body and the hydrated support coalescing without bilayerformation.

The bilayer may be visualised through the hydrated support with aninverted microscope or in some circumstances even by the naked eye. Thevisualisation of the lipid bilayer may be used to track the formation,position, size, or other property of the bilayer. Visualisation of thebilayer allows labelled analytes/proteins/compounds at or in the bilayerto be seen and studied.

The bilayer of amphipathic molecules may be used to study processesoccurring at, in or through the bilayer. The bilayer can be used as anartificial/model system in which to study cell membrane behaviour.

Proteins may be inserted into the bilayer of amphipathic molecules.

Proteins in the environment of the bilayer, for example in thehydrophobic medium and/or in the hydrophilic body and/or in the hydratedsupport, may insert spontaneously into the bilayer. Alternativelyproteins may be driven into the bilayer by the application of a voltageand/or by fusion of protein loaded vesicles with the bilayer. Thevesicles may be contained within or introduced to the hydrophilic body.Proteins may be introduced into the membrane by using the probe methoddisclosed in GB0614835.7. Proteins may insert into the bilayer in thesame manner as if the bilayer was formed by known techniques.

The inserted protein may be a known membrane-associated protein.

The protein may be a membrane-associated protein which is anchoreddirectly or indirectly to the bilayer. The protein may be a selective ornon-selective membrane transport protein, an ion channel, a pore formingprotein or a membrane-resident receptor.

Membrane-associated proteins which may associate with and/or insert intothe bilayer include any of the group comprising peptides, e.g.gramicidin; α-helix bundles, e.g. bacteriorhodopsin or K⁺ channels; andβ-barrels, e.g. α-hemolysin, leukocidin or E. coli porins; orcombinations thereof.

The bilayer of amphipathic molecules may be used to detectcompounds/analytes which are capable of interaction with amphipathicmolecules in the bilayer or with a membrane-associated protein in thebilayer. The interaction with the membrane-associated protein or theamphipathic molecules may be by the specific or non-specifictranslocation of the analytes/compounds across the bilayer, this may bemediated by the membrane-associated protein or by the amphipathicmolecules. Alternatively compounds/analytes may interact with atrans-membrane protein or with the lipid bilayer to cause physical,optical, electrical, or biochemical changes. Such interaction may bedetected in many different ways, including, but limited to, by visualchanges, changes in specific capacitance, or by the activation offluorescently labelled lipids or proteins in the bilayer.

The bilayer may be used to detect membrane-associated proteins.Preferably the membrane-associated proteins are ion channel proteinsand/or pore forming proteins. Preferably the membrane-associatedproteins diffuse into and/or associate with the bilayer causing adetectable change in the properties at the bilayer. The propertieschanged may be physical, optical, electrical or biochemical.

Bilayers of amphipathic molecules made by any method of the inventionmay be used to investigate and/or screen membrane-associated proteins;to investigate and/or screen for analytes that interact withmembrane-associated proteins; and to investigate and/or screen forcompounds that interact with bilayers made of different amphipathicmolecules. Bilayers of different amphipathic molecules may be screenedto study the role of and/or the interaction of different amphipathicmolecules with various analytes/compounds.

The bilayer may be used to study the voltage dependence properties of amembrane-associated protein inserted in the bilayer. For example, abilayer composed of DPhPC may have a specific capacitance between about0.3 and about 0.9 μF cm⁻² at 22° C., preferably about 0.65 μF cm⁻² at22° C. By studying changes in the specific capacitance properties thechanges in the properties of the bilayer caused by different conditionscan be studied. For example, by studying the ionic current crossing abilayer the properties of an ion permeable transmembrane-associatedprotein may be studied, as may the interaction of thetransmembrane-associated protein with different analytes/compounds.

The bilayer may be used to study the ability of a membrane-associatedprotein inserted in the bilayer to transport molecules across themembrane. For example, the amount of a molecule being transported acrossa membrane, either through a protein or simply non-mediated transportacross the bilayer, may be determined by using voltage studies, massspectrometry, enzymatic techniques, such as ELISA, or by using afluorescently or radioactively labelled substrate.

A bilayer produced by any method of the invention may also be used tostudy the effects of mechanical changes of the bilayer on proteins inthe bilayer or on the bilayer itself. Mechanical changes which can bestudied include, for example, changes in membrane curvature, lateralforces, surface tension etc.

A compound/analyte to be tested, studied and/or used in a screen may beintroduced into the system by placing it in the hydrophilic body and/orin the hydrated support and/or in the hydrophobic medium. If included inthe hydrophilic body the compound/analyte may be incorporated when thebody is formed or it may be added later, for example, by injection intothe formed body. Similarly, if the compound/analyte is in the hydratedsupport it may be incorporated when the support is formed or addedlater.

In one embodiment the hydrated support may be a protein separation gelor membrane containing proteins (analytes/compounds) for analysis. Forexample the sample may be a polyacrylamide gel containing proteins whichhave been separated on the basis of size. In this embodiment theanalytes/compounds are introduced to the bilayer via the hydratedsupport.

The analyte/compound may be a purified protein or a crude proteinextract.

The analytes/compounds to be tested may be in a sample. The sample maybe an environmental sample, for example from a body of water such as ariver or reservoir, or the sample may be a biological sample, forexample a sample of blood, urine, serum, saliva, cells or tissue. Thesample may be a liquid from a cell growth medium.

One or more detection means may be used to detect chemical, biochemical,electrical, optical, physical and/or environmental properties of thebilayer of amphipathic molecules or of membrane-associated proteinsinserted into the bilayer. In particular the one or more detection meansmay be used to detect changes in or at the bilayer induced by thecompounds/analytes.

The detection means may comprise electrodes which may be used to detectchanges in ionic current passing through a protein channel inserted intoa bilayer or the electrochemical properties of molecules in the hydratedsupport, hydrophilic body or bilayer.

Chemical or biochemical changes may be detected using enzymatic assaysor immunoassays. Alternatively, the use of labelled, for example radioor fluorescently labelled, proteins which are activated under certainconditions can be used to monitor changes at the bilayer. Colormetricmethods that respond to changes in light absorption upon reaction mayalso be used to detect changes in the bilayer, in particular this methodmay be used to detect a change in the size of the bilayer.

The detection means may be capable of constantly or intermittentlydetecting properties of, or changes at, the bilayer.

Detection reagents, like membrane-associated proteins, analytes andother compounds may be delivered to the bilayer by incorporation intothe hydrophilic body and/or the hydrated support, injection directlyinto the hydrophilic body and/or the hydrated support, and/orincorporation in, or addition to, the hydrophobic medium. Injection intothe hydrophilic body and/or the hydrated support may be achieved using amicropipette.

In an embodiment where changes in membrane capacitance are being studiedelectrodes may be used as the detection means. The electrodes may beAg/AgCl, such electrodes may be from approximately 10 microns to 1 mm indiameter. A first electrode may be electrically contacted with thehydrophilic body and a second electrode may be electrically contactedwith the hydrated support. Electrical properties of the bilayer, such asthe specific capacitance of the bilayer, may be determined using theelectrodes.

A micromanipulator may be used to insert electrodes into the hydrophilicbody and/or the hydrated support.

The physical, chemical or electrical environment of the bilayer may becontrolled by the introduction, removal, or sequestering of reagents,analytes, compounds and/or proteins to or from the bilayer and/orhydrophilic body and/or hydrated support, e.g. the pH of the environmentsurrounding the bilayer may be controlled by the addition of a buffer tothe hydrophilic aqueous body and/or the hydrated support.

The bilayer may be repeatedly reformed, by removing contact between thehydrophilic body and the hydrated support and then re-establishingcontact to re-form the bilayer. The bilayer may be disassembled byincreasing the distance between the centre of the hydrophilic body andthe hydrated support to a point where the bilayer would become unstableand spontaneously disassemble.

Membrane-associated proteins in the bilayer may be removed fromassociation with the bilayer by disassembling the bilayer for example byremoving contact between the hydrophilic body and the hydrated support.Once the bilayer has been disassembled to remove the membrane-associatedproteins, it may be reformed such that no membrane-associated proteins,or different membrane-associated proteins, are associated with thebilayer.

The concentration of membrane-associated proteins associated with thebilayer may be increased by decreasing the area of the bilayer.Conversely, the concentration of membrane-associated proteins associatedwith the bilayer may be decreased by increasing the area of the bilayer.

The ability to increase the concentration of membrane-associatedproteins in the bilayer may be used to produce 2D crystals ofmembrane-associated proteins by decreasing the area of the bilayercomprising the membrane-associated proteins and hence restricting thearea within which a membrane protein can diffuse.

The ability to increase or decrease the concentration ofmembrane-associated proteins in the bilayer may be used to modulateprotein-protein interactions, for example between sub-units of a proteinor between components of a protein complex.

Once formed, a bilayer made according to any method of the invention maybe translocated or moved across the surface of the hydrated support.Preferably this is achieved by moving the hydrophilic body across thesurface of the hydrated support. The lipid bilayer may be translocatedacross the surface of the hydrated support at speeds of about 1 or 2 mms⁻¹ or more. Preferably membrane-associated proteins within the bilayerdo not disassociate from the bilayer when the bilayer is translocatedacross the surface of the hydrated support.

The translocation of the bilayer across the surface of the hydratedsupport may be achieved by moving a member contacted with thehydrophilic body or the hydrated support. Preferably the member is anelectrode. Preferably a micromanipulator is used to move the member inorder to translocate the bilayer by moving either the hydrophilic bodyacross the hydrated support or the hydrated support across thehydrophilic body. Alternatively both the hydrated support and thehydrophilic body may move.

The translocation of the bilayer may be used to apply forces to proteinsin the bilayer, for example to study mechano-sensitive protein channelsor to study the effect of such force on the properties of the bilayeritself.

The translocation of the bilayer may be used to scan across the surfaceof the hydrated support to identify analytes/compounds in the hydratedsupport which alter properties of the bilayer and/or membrane-associatedproteins in the bilayer.

The surprising capability of the bilayer to translocate across thesurface of the hydrated support provides the benefit of being able toscan across the hydrated support with the bilayer to detectanalytes/compounds, such as membrane-associated proteins and/or reagentsor substrates located at different regions of the same hydrated support.This advantageously can be done without having to disassemble thebilayer between each sampling region.

The bilayer may be translocated across a hydrated support comprising anarray or library of different compounds. The different compounds may bespotted onto the support in predetermined positions. Alternatively thehydrated support may comprise a separation gel or membrane, containingcompounds such as proteins or DNA, that have been separated on the basisof their size or ionic properties.

The translocation of one or more bilayers may allow a bilayer comprisingone or more particular membrane-associated proteins to be rapidlyscreened against a library of compounds in the hydrated support. Thescreen may allow compounds in a library which interact with amembrane-associated protein and cause a detectable change in propertiesat the bilayer to be detected. The compounds in the library may beproteins, DNA or other small molecules. The detectable change may be,for example, a change in conductance or a change in fluorescence orother marker pattern.

Alternatively, or additionally, translocation of the bilayer may allow alibrary of compounds in a hydrated support to be screened for potentialmembrane-associated proteins. Again, the membrane-associated proteinsmay be detected by a change in properties at the bilayer, for example, achange in conductance or capacitance across the membrane. The librarymay comprise protein extracted from a cell or a population of cells.

The bilayer may be formed on a porous hydrated support which may then bescanned across the surface of a cell to detect local concentrationdifferences in excreted compounds, such as ATP. The cell may beprokaryotic or eukaryotic. The bilayer may be used to detectanalytes/compounds, such as cell excretions, on a cell surface in vitroor in vivo.

The bilayer may be formed on a suspended hydrated support in a mobiledevice, such as a pipette tip, which may then be scanned across thesurface of a cell. Alternatively the mobile device may be used to probedifferent solutions, for example, different biological samples.

A plurality of separate bilayers may be formed between a plurality ofseparate hydrophilic bodies and one or more hydrated supports. Ahydrophilic body may be contacted with one or more other hydrophilicbodies on the same hydrated support to form a plurality of separatelipid bilayers between each of the hydrophilic bodies as well as betweenthe hydrophilic bodies and the hydrated support. The hydrophilic bodiesmay be arranged in a two, or potentially three, dimensional array.

Two or more separate hydrophilic bodies on the same hydrated support maycomprise the same or different detection means and/or differentreagents, and/or the same or different membrane-associated proteinsrelative to each other.

An array of aqueous droplets may be deposited over a hydrated supportsurface to detect the location of analytes/compounds in the hydratedsupport, for example by the fluorescence of a hydrophilic body or achange in recorded conductance when an analyte/compound is detected.

According to another aspect the invention provides a bilayer productcomprising a hydrophilic body and a hydrated support with a bilayer ofamphipathic molecules therebetween. Preferably the bilayer is formed byinteraction of a monolayer of amphipathic molecules on the hydrophilicbody and a monolayer of amphipathic molecules on the hydrated support.

The bilayer may be formed by any method of the invention.

The skilled man will appreciate that all the preferred featuresdiscussed with reference to the first or second aspect of the invention,and in particular those relating to the bilayer itself and not itsproduction, can be applied to a bilayer product according to theinvention and to all aspects of the invention which use a bilayer.

According to another aspect of the invention, there is provided the useof a bilayer product according to the invention in conjunction withfluorescence microscopy.

The nature of the hydrated support preferably allows the bilayer to beviewed using a microscope. Thus in this aspect of the invention thehydrated support preferably is a layer no more than about 2 mm thick.Preferably the hydrated support is from about 1 nm to about 2 mm thick,preferably from about 100 nm to about 1 mm thick, more preferably thehydrated support is from about 100 nm to about 400 nm thick.

Preferably when a thin hydrated support of less than about 400 nm thickis used it is kept in contact with a re-hydrating medium, such as alarger bulk of hydrating liquid or hydrated support material, to preventdrying out of the thin support material. The rehydrating medium may bethe same or different in material/composition to the thin hydratedsupport, and is present to prevent the thin layer from dehydrating. Therehydrating support may be agarose gel, water or polyacrylamide gel. Therehydrating support may be porous or solid.

Molecules to be observed may be fluorescently labelled withfluorophores.

Preferably fluorophores in the hydrophilic droplet and/or hydratedsupport are observed using total internal reflection fluorescence.

Preferably observations using total internal reflection fluorescence areused in combination with electrical measurements.

Total internal reflection fluorescence microscopy may be used to observefluorescence from entities present in the bilayer either as a bulkproperty of the bilayer, or with suitable detection down to the level ofindividual molecules.

The advantage of using total internal reflection fluorescence to observethe fluorophores is that only fluorophores within about 200 nm of thelipid bilayer are illuminated and thus observed, whilst otherfluorophores not close to the lipid bilayer are not illuminated and notobserved. Using total internal reflection fluorescence measurements incombination with electrical measurements has the advantage thatprotein-protein interactions can be studied, for example, the assemblyof channel proteins such as α-hemolysin from labelled subunits or theelectrical response of ion channels to the binding of a fluorescentsubstrate can be studied.

According to a yet further aspect of the invention there is provided amethod of screening for an interaction between a bilayer of amphipathicmolecules and one or more compounds in a library comprising:

-   -   i) providing a bilayer product comprising a hydrophilic body and        a hydrated support with a bilayer of amphipathic molecules        therebetween;    -   ii) translocating the hydrophilic body and thus the bilayer        across the surface of the hydrated support; and    -   iii) detecting any interaction between the bilayer and a        compound in the hydrated support.

Preferably the bilayer product is made by the method of the first orsecond aspect of the invention.

Preferably the hydrated support comprises the library of compounds to betested.

In one embodiment a membrane-associated protein may be inserted into thebilayer before or as the bilayer is translocated across the hydratedsupport.

Translocation of the bilayer may be achieved by the direct or indirectcontact of the hydrophilic body with a micromanipulator arranged to movethe hydrophilic body. The hydrophilic body may be in contact with anelectrode which may be moved to translocate the bilayer across thehydrated support.

The method of screening may be automated to allow high throughputscreening to be undertaken.

The skilled man will appreciate that the preferred features of anyaspect of the invention relating the method of producing a bilayer, to abilayer per se and to the translocation of a bilayer can be applied tothis aspect of the invention.

According to a further aspect of the invention, there is provided theuse a bilayer product according to the invention to identify one or moremembrane-associated protein present in and/or on the hydrated support orthe hydrophilic body.

According to another aspect of the invention, there is provided the useof a bilayer product according to the invention to identify substancescapable of interaction with a membrane-associated protein located in thebilayer.

According to another aspect of the invention, there is provided a methodfor detecting one or more analytes present in an aqueous solutioncomprising the steps of:

-   -   (a) providing a lipid-in-oil solution in a walled vessel,        wherein at least part of a wall of the vessel comprises a porous        hydrated support;    -   (b) providing a hydrophilic body in the lipid-in-oil solution;    -   (c) forming a first monolayer of lipid molecules on the surface        of the porous hydrated support and a second monolayer of lipid        molecules on the surface of the hydrophilic body;    -   (d) contacting the porous hydrated support with the hydrophilic        body such that a lipid bilayer forms between the lipid monolayer        on the hydrophilic body and the lipid monolayer on the porous        hydrated support;    -   (e) contacting the porous hydrated support with the aqueous        solution such that the analytes present in the aqueous solution        are available to the lipid bilayer;    -   (f) detecting the insertion of analytes into the lipid bilayer        and/or the translocation of the analytes across the lipid        membrane and/or the interaction of analytes with the bilayer,        using detection means.

An apparatus for use with a lipid bilayer comprising a walled vessel forretaining a lipid/oil solution, wherein at least one portion of a wallof the vessel comprises a porous support arranged to be hydrated in use.

The apparatus may comprise a detection means. Preferably the detectionmeans is an electrode or a photodetector.

Preferably the apparatus is portable. Preferably the apparatus ishandheld.

The walled vessel may be a pipette tip.

It will be appreciated that all the optional and/or preferred featuresof the invention discussed in relation to only some aspects of theinvention can be applied to all aspects of the invention.

Preferred embodiments/aspects of the invention will now be described byway of example only with reference to the accompanying figures, inwhich:

FIG. 1—illustrates an aqueous droplet (hydrophilic body) on a hydratedsupport bilayer, referred to herein in as a droplet-on-hydrated-supportbilayer (DHB); FIG. 1A is a diagram of a droplet-on-hydrated-supportbilayer. A lipid monolayer spontaneously forms on the aqueous surface ofthe aqueous (water) droplet and the hydrated support (hydrogel) wheneach is immersed in a solution of lipid in hydrophobic oil. When themonolayers of the two components are brought into contact they form alipid bilayer; FIG. 1B shows a droplet bilayer (DHB) visualised frombelow with an inverted microscope—the image shows a droplet, without anelectrode, supported on a hydrogel surface. The single continuousbilayer area in the centre of the droplet is easily seen due to thelarge change in refractive index at the interface; FIG. 1C demonstratesthat the lipid bilayer between the droplet and hydrated support can beelectrically accessed via electrodes inserted into both the droplet andthe hydrated support. The electrical capacitance trace shows theformation of a lipid bilayer. Bilayer capacitance is determined byapplying a triangular potential waveform to the lipid bilayer andmeasuring the square wave peak-to-peak current response;

FIG. 2—illustrates the scanning of proteins in hydrogels using adroplet-on-hydrated-support bilayer; FIGS. 2A and B show compositeimages of polyacrylamide gels after droplet-on-hydrated-support bilayerscanning, created by overlaying an image of the dried gel (containingvisible pre-stained maker lane M) with autoradiographs to visualise theradio-labelled protein bands. Marker lane (M) bands correspond tomolecular weights of approximately 210, 111, and 71 kDa; FIG. 2A is anSDS-PAGE gel containing the potassium channel Kcv; FIG. 2B is anSDS-PAGE gel containing the pore forming protein alpha hemolysin (aHL)aHL-WT (WT) and aHLM113F-D8 (113F) which forms heptameric protein pores.After immersion in DPhPC/C16 (lipid/oil) solution the gels were scannedwith 200 mL aqueous droplets which had formed a bilayer on contact withthe gel (hydrated support). Protein insertion and binding at the bilayerwas monitored via patch-clamp amplified electrical recordings as afunction of droplet-bilayer position on the gel surfaces; FIG. 2C showstypical electrical recordings from the regions of the gels containingKcv (+20 mV, 500 mM KCl, 10 mM Hepes, pH 7.0), αHL-WT and αHL-M113F-D8(+10 mV, 1 M KCl, 10 mM Na₁PO₄, pH 7.0). The aHL channels were scannedwith droplets containing 10 μM β-cyclodextrin (βCD) to differentiatebetween the two mutants; FIG. 2D shows that channel-protein insertionwas only observed in localised regions about the separated proteinbands. This is illustrated by a 12 mm linear scan across the aHL-WT band(dotted line marked on gel in FIG. 2B), which shows the rate ofchannel-protein insertion as a function of bilayer position;

FIG. 3—illustrates the results of scanning cell extracts in gels usingdroplet-on-hydrated-support bilayers; FIG. 3A shows a coomassie stainedpolyacrylamide gel after droplet-on-hydrated-support bilayer scanning,showing SDS-PAGE purified E. coli cell extracts. The E. coli cell lineswere separately transformed to produce αHL-WT (lane 1) and αHL-M113F-D8(lane 2) through leaky expression. After immersion in DPhPC/C, solutionthe gel was scanned with 200 mL droplets containing 10 μMβ-cyclodextrin. Protein insertion and binding in the bilayer wasmonitored via patch clamp amplified electrical recordings as a functionof droplet-bilayer position on the gel surface; FIG. 3B shows typicalelectrical recordings from scans of the regions circled on the gel.Large numbers of αHL-WT (top) and αHL-M113F-D8 (middle) channelsinserted from the regions indicated. Small numbers of unidentifiedporin-like channels (bottom) were found in the lower region of the gel;

FIG. 4—illustrates scanning of cyclodextrins in gels usingdroplet-on-hydrated-support bilayers; FIG. 4A shows a schematic of theexperimental approach employed to scan molecules doped intopolyacrylamide gels by absorption. γ-cyclodextrin (γCD) and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (hβCD) were introduced to the bottomof the gel approximately 10 mm apart. After immersion and stabilisationin the lipid/oil solution the gel was scanned with droplets containingαHL-WT channels. Cyclodextrin binding to αHL-WT channels in the dropletbilayer was monitored via patch clamp amplified electrical recordings asa function of droplet-bilayer position on the gel surface; FIG. 4B showsthe binding characteristics of γCD (top, 68% block) and hβCD (bottom,95% block) to aHL-WT are clearly distinguishable in electricalrecordings (−50 mV, 1 M KCl, 10 mM Na₁PO₄, pH 7.0); FIG. 4C shows thebinding frequency of the two cyclodextrins plotted as a function ofdistance in a scan between the two doped locations. Lines indicateGaussian fits to the measured binding frequency;

FIG. 5—shows a diagram of a droplet (hydrophilic body) ejected from apipette tip, whilst immersed in a lipid/oil solution. The droplet iscontacted with a hydrogel (hydrated support) to form a lipid bilayer;

FIG. 6—illustrates a droplet-on-hydrated-support bilayer produced usingthe method of FIG. 5 visualised from below using an inverted microscope;

FIG. 7—shows a diagram of a lipid bilayer formed on a hydrated supportfixed to a tip of a pipette which can be used to scan or probe anaqueous system;

FIG. 8—shows a diagram of how the lipid bilayer can be increased anddecreased in area by moving the centre of the aqueous droplet towards oraway from the hydrated support using an electrode inserted in thedroplet or in electrical contact with the droplet;

FIG. 9—illustrates how a second aqueous droplet carrying a cargo ofreagents can be burst together with an aqueous droplet without rupturingthe bilayer;

FIG. 10—shows a diagram of how reagents can be introduced into anaqueous droplet by injection from (A) a micro-pipette, or (B) amicro-pipette with a multi bore capillary;

FIG. 11—illustrates multiple droplets-on-hydrated-support bilayers on asingle hydrated support. FIG. 11A—shows independent/separate droplets;FIG. 11B—shows connected droplets forming multiple lipid bilayersbetween the droplets and with the underlying hydrated support;

FIG. 12—illustrates total internal reflection fluorescence microscopy ona droplet-on-hydrated-support bilayer;

FIG. 13A—shows an alternative portable device for use in producing abilayer according to the invention;

FIG. 13B—illustrates a bilayer produced using the device of FIG. 13Avisualised from below using an inverted microscope;

FIG. 14A—shows a device for producing an array of bilayers;

FIG. 14B—illustrates a bilayer produced using the device of FIG. 14A;

FIG. 15A—shows a device for producing a bilayer in which the aqueousphase is capable of perfusion;

FIG. 15B—illustrates electrical traces produced using the device of FIG.15A, in the top trace α-hemolysin channels are shown inserting into thebilayer, in the bottom trace cyclodextrins are shown binding;

FIG. 15C—illustrates a bilayer produced by the device of FIG. 15A;

FIG. 16—shows an alternative device to that of FIG. 15A in which thebulk aqueous volume is a microfluidic channel;

FIG. 17—illustrates a large bilayer produced by the method of theinvention;

FIG. 18—illustrates a further large bilayer produced by the method ofthe invention; and

FIG. 19—illustrates the concentration of membrane bound proteins using abilayer according to the invention.

“A droplet-on-hydrated-support bilayer” as referred to in the specificexamples is the same as “a bilayer of amphipathic molecules” aspreviously discussed.

METHODS

1,2-Diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids),hexadecane (Sigma-Aldrich), β-cyclodextrin (Sigma-Aldrich), andγ-cyclodextrin (Sigma-Aldrich), heptakis(2,3,6-tri-o-methyl)-β-cyclodextrin (Cyclolab) were used without furtherpurification.

In Vitro Transcription/Translation of Proteins

αHL-WT, αHL-M113F-D8 (Gu, L. Q. et al., 2001. Journal of GeneralPhysiology 118, 481-493) (RL2 background (Cheley, S. et al., 1999.Protein Science 8, 1257-1267) and a C-terminal D8 extension to produce agel shift relative to αH-WT) and Kcv were prepared from genes cloned inthe pT7.SC1 vector (Cheley, S. et al., 1997. Protein Engineering 10,1433-1443) using a coupled in vitro transcription/translation (IVTT) kit(Promega Corporation) as previously described (Walker, R. et al., 1992.Journal of Biological Chemistry 267, 10902-10909). ³⁵S-methionine wasincorporated into the proteins for visualisation by autoradiography. 50μL in vitro transcription/translation reactions of the aHL proteins wereoligomerised as described previously (Walker, B. et al., 1992. Journalof Biological Chemistry 267, 10902-10909), then pelleted and resuspended(20 μL, 10 mM MOPS buffer, pH 7.4, 150 mM NaCl, 1 mg mL⁻¹ BSA). Prior toelectrophoresis, the 20 μL resuspended oligomer samples were mixed with5 μL of 5×SDS-containing Laemmli buffer (final concentration: 10% (v:v)glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 0.0625 M Tris, pH 7.5).

100 μL in vitro transcription/translation reactions were performed forKcv, and the products were subsequently separated in a 10% Tris-HCl gelby electrophoresis. The gel was dried onto paper under vacuum at roomtemperature and then imaged by autoradiography. The band correspondingto the Kcv tetramer was cut from the gel and rehydrated (300 μl, 10 mMHepes, pH 7.4). The rehydrated gel was crushed and transferred to a 0.2μm cellulose acetate microfiltration tube (Rainin) and centrifuged at25000 g for 30 minutes to recover the solubilised protein.

Electrophoresis of In Vitro Transcription/Translation Proteins

5 μL aliquots of the gel-purified Kcv tetramers were loaded into 8.5%Tris-acetate polyacrylamide gels and subjected to electrophoresis (200V, 20 minutes) in TBE buffer (8.9 mM Tris, pH 8.3, 8.9 mM boric acid,200 μM EDTA, 0.1% SDS) to separate the protein bands. The gel tank wasthen refilled with SDS-free TBE buffer, and electrophoresis wascontinued (50 V, 2 hours) to remove SDS in the gel.

5 μL aliquots of the in vitro transcription/translation α-hemolysinoligomers were loaded into 7% Tris-acetate polyacrylamide gels (XTCriterion; Bio-Rad Laboratories Inc.) and subjected to electrophoresis(200 V, 1 hour) in XT Tricine buffer (Bio-Rad Laboratories Inc.) toseparate the protein bands. The gel tank was then refilled with SDS-freeLaemmli buffer, and electrophoresis was continued (100 V, 2 hours) toremove SDS.

All gels were run with a pre-stained marker lane (SeeBlue Plus2,Invitrogen). Following droplet-on-hydrated-support bilayer gel scanningthe gels were imaged by autoradiography.

E. coli Crude Extraction and Electrophoresis

Competent E. coli JM109(DE3) cells (Promega Corporation) weretransformed by heat-shock with pT7-plasmids encoding either αHL-WT orαHL-M113F-D8. Single colony transformants were picked and cultured for16 hours in 2 mL LB-medium containing 50 μg mL-1 ampicillin. The cellswere then centrifuged at 2500 g for 20 minutes and resuspended (200 μL,25 mM MOPS, pH 7.4, 150 mM NaCl, 0.5% (w:v) SDS, 500 ng DNase 1).Following 30 minutes of incubation on ice the 200 μL samples were mixedwith 50 μL of 5×SDS-containing Laemmli buffer (final concentration: 10%(v:v) glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 0.0625 M Tris, pH 7.5).45 μL of this solution was then loaded into 10% Bis-Tris polyacrylamidegels (XT Criterion; Bio-Rad Laboratories Inc.) and subjected toelectrophoresis (200 V, 30 minutes) in XT MOPS buffer (Bio-RadLaboratories Inc.). The gel tank was then refilled with SDS-free buffer(50 mM MOPS, 50 mM Bis-Tris, pH 7.0), and electrophoresis was continued(100 V, 2 hours) to remove SDS.

Following droplet-on-hydrated-support bilayer gel scanning the gels werestained with Coomassie Brilliant Blue (Sigma-Aldrich).

Droplet-on-Hydrated-Support Bilayer Gel Scanning

After electrophores the Kcv gels were immersed in 10 mM Hepes buffer (pH7.0) containing 500 mM KCl for at least 30 minutes. The aHL and E. coligels were immersed in 10 mM Na₂PO₄ (pH 7.0) buffer containing 1M KCl.After dialysis the gels were left in 10 mM DPhPC/C₁₆ solution for 15minutes, then scanned with approximately 200 nL droplets of the samebuffer as that in the gel. Droplets were moved about the surface of thehydrogels with the inserted Ag/AgCl electrode attached to a dxdydzmicromanipulator (NMN-21; Narishige).

Electrical Measurements and Bilayer Imaging

100 μm diameter Ag/AgCl wire electrodes were used to electrically accessthe droplets and gels. Currents were recorded with a patch clampamplifier (Axopatch 200B; Axon Instruments), and digitized at 1 kHz(MiniDigi-1A; Axon Instruments). Electrical traces were filteredpost-acquisition (100 Hz lowpass Gaussian filter) and analyzed usingpClamp 9.0 software (Axon Instruments). The gel scanning apparatus andamplifying headstage were enclosed in a Faraday cage attached to aninverted microscope (TE-2000; Nikon Instruments UK) equipped with acamera (DS-1QM; Nikon) for imaging and positional tracking of thedroplet-on-hydrated-support bilayers.

Results Creating Droplet-on-Hydrated-Support Bilayers

10 mM 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in hexadecane(C₁₆) was used as the lipid/oil solution in all experiments. Aqueousvolumes immersed in this solution spontaneously self-assemble a DPhPCmonolayer, and when the monolayers of two components are brought intocontact they spontaneously form a lipid bilayer (Tsofina, L. M. et al.,1966. Nature 212, 681-683; Malmstadt, N. et al., 2006. Nano Letters 6,1961-1965; Funakoshi, K. et al., 2006. Analytical Chemistry 78,8169-8174; Holden, M. A. et al., 2007. Journal of the American ChemicalSociety—in press). A droplet-on-hydrated-support bilayer is formed bycontacting aqueous droplets with porous hydrated supports such ashydrogels (FIG. 1A). A stabilisation period of at least 15 minutes wasrequired before contacting monolayers to prevent fusion. After thisperiod, bilayer formation was observed with almost 100% efficiencywithin a few seconds to a minute of contact of an aqueous droplet withthe hydrated support. The droplet-on-hydrated-support bilayers werevisualised on an inverted microscope (FIG. 1B), this technique was usedto track the position of a lipid bilayer during experiments.

The lipid bilayers were electrically accessed by inserting a 100 μMdiameter Ag/AgCl electrode into the droplets (FIG. 1A) using amicromanipulator. With a corresponding Ag/AgCl ground electrode in thehydrated support, electrical measurements across the lipid bilayer werecarried out. The lipid bilayers were typically able to withstandvoltages up to approximately 300 mV while retaining seals in excess of100 GΩ. Electrical noise levels were typically of the order of ±0.5 pArms with a 1 kHz recording bandwidth. This reflects the limitations ofthis apparatus and not the inherent noise in droplet-on-hydrated-supportbilayers.

Synchronous optical measurements of bilayer area (FIG. 1B) inconjunction with capacitance measurements (FIG. 1C) yielded a specificcapacitance of 0.65 μF cm⁻² at 22° C. for the DPhPC bilayers in thissystem. This agrees well with other reported values (0.4 to 0.8 μF cm⁻²)(Montal, M. & Mueller, P. 1972. Proceedings of the National Academy ofSciences of the United States of America 69, 3561-3566; Fujiwara, H. etal. 2003. Journal of Chemical Physics 119, 6768-6775; Funakoshi, K. etal., 2006. Analytical Chemistry 78, 8169-8174), indicating the lipidbilayers are similar in thickness to their planar bilayer counterparts.

Gel Scanning with Droplet-on-Hydrated-Support Bilayers

By scanning the position of a bilayer across a gel while makingsingle-channel recording measurements it is possible to resolve thelocation of isolated membrane channels as they insert into the lipidbilayer. When applied to electrophoretically separated protein bands inhydrogels, scanning allows the determination of whether a particularband contained a channel-forming protein. To validate this technique,polyacrylamide gels containing the viral potassium channel Kcv (Plugge,B. et al., 2000. Science 287, 1641-1644; Gazzarrini, S. et al., 2003.Febs Letters 552, 12-16), and two mutants of the staphylococcal poreforming toxin α-hemolysin (αHL) (Song, L. Z. et al., 1996. Science 274,1859-1866) were scanned.

SDS-PAGE was used to separate in vitro transcription/translation (IVTT)expressed Kcv, wild-type α-hemolysin (αHL-WT) and a M113F α-hemolysinmutant (αHL-M113F-D8) (Gu, L. Q. et al., 2001. Journal of GeneralPhysiology 118, 481-493) in polyacrylamide gels (FIG. 2A and FIG. 2B).SDS-PAGE was followed by electrophoretic cleaning to remove free SDSfrom the gels (which would otherwise destabilise the lipid bilayer).This step can be omitted when running gels under native detergent-freeconditions. After electrophoresis, the gels were dialysed in appropriatebuffers containing KCl to introduce the electrolyte necessary forelectrical recordings. Following dialysis the gels were immersed in theDPhPC/C₁₆ lipid/oil solution for 30 minutes, and then scanned with 200nL droplets (producing bilayers of approximately 200 μm in diameter).The droplets were moved about the surface of the hydrogels bytranslating an inserted Ag/AgCl electrode, and the lipid bilayerposition was tracked visually on an inverted microscope.

When droplet-on-hydrated-support bilayers were positioned over regionsof the gels containing channel proteins stepwise changes in ion-currentresulting from the spontaneous insertion of channels could be detected.FIG. 2C shows typical examples of electrical traces acquired whenscanning the respective regions of the gels containing Kcv, αHL-WT andαHLM113F-D8. Kcv behaviour is characterised by stepwise bursts ofcurrent as the channels transiently open and close (FIG. 2C top). αHL-WTpores remain open, resulting in stepwise increases in current for eachinsertion event (FIG. 2C middle).

To demonstrate the ability to differentiate between the two α-hemolysinmutants the αHL gels were scanned with droplets containingβ-cyclodextrin (βCD). β-cyclodextrin acts as a non-covalent blocker thatlodges inside the β-barrel of αHL, which is observed as a reversiblestepwise change in current in an electrical recording (Gu, L. Q. et al.,1999. Nature 398, 686-690; Gu, L. Q. & Bayley, H. 2000. BiophysicalJournal 79, 1967-1975). αHL-WT does not bind β-cyclodextrin strongly(Gu, L. Q. et al., 1999. Nature 398, 686-690; Gu, L. Q. & Bayley, H.2000. Biophysical Journal 79, 1967-1975), whereas the aHL-M113F-D8mutant binds β-cyclodextrin strongly with a voltage-dependent mean dwelltime of approximately 10 seconds (Gu, L. Q. et al., 2001. Journal ofGeneral Physiology 118, 481-493). Without β-cyclodextrin the electricalcharacteristics of the two aHL variants are essentially identical. Withβ-cyclodextrin the αHL-M113F-D8 channels are easily distinguishable bythe β-cyclodextrin binding events overlaying the stepwise increases inconductance (FIG. 2C bottom).

It was found that during gel scanning the proteins did not appear todiffuse, and insertion events were only observed in highly localisedregions about the focused bands in the gel. This is illustratedquantitatively in FIG. 2D, which shows protein insertion rate in alinear scan across the wild-type αHL band using a droplet with a lipidbilayer of approximately 200 μm in diameter.

The gel scanning experiments where extended to SDS-PAGE purified cellextracts. FIG. 3 shows the results of scanning a SDS-PAGE gel (FIG. 3A)containing crude extracts from E. coli, transformed to produce αHL-WT(lane 1) and αHL-M113F-D8 (lane 2) through leaky expression. As with theprevious gel example in FIG. 2, these proteins could be electricallycharacterised (FIG. 3B) from the expected regions of the gel as shown bysubsequent Coomassie staining. Channel insertion rates were higher thanobserved from the in vitro transcription/translation gels, reflectingthe substantially higher concentrations of protein produced fromexpression in E. coli. Surprisingly, in addition to aHL channels anumber of other channel proteins were detected with markedly differentbehaviour (e.g. FIG. 3B bottom), again localised to specific areas ofthe gel. The channels typically insert in multiples of three and showsubstantial voltage-dependent gating behaviour.

It was found that extended immersion in the lipid/oil solution duringgel scanning does not result in any substantial loss of protein from thegel matrix. Similarly, extended immersion in electrolyte buffer duringthe dialysis step does not noticeably deplete the proteins in the gel.As a result, individual gels can be re-used in many, at least six,consecutive gel scanning experiments, and the gel buffer conditions canbe varied as required. Furthermore, the scanning procedure does notaffect the ability to subsequently stain or image the gel, or to recoverspecific proteins from the gel for further analysis.

Analyte Detection with Droplet-on-Hydrated-Support Bilayers

Essentially reversing the gel scanning experiment, protein channels indroplets on hydrated support bilayers can be used as molecular sensorsto scan different analytes within hydrogels.

2% polyacrylamide gels were doped via absorption from blotted proteinsolution with approximately 10 μM of γ-cyclodextrin (γCD) andheptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (hβCD) in two regions spacedapproximately 10 mm apart (FIG. 4A). Following a 30 minute immersion ina DPhPC/C₁₆ solution, the gel was scanned between the two cyclodextrinregions with a 200 mL droplet containing αHL-WT. Under the experimentalconditions γCD binding to αHL-WT produces a current block of 68%, andhβCD binding to aHL-WT produces a current block of 95%. These differentbinding amplitudes permit positive identification of both analytes withaHL-WT (FIG. 4B).

In this experiment the droplet was translated across the hydrogelwithout removing the lipid bilayer from the surface, retaining theαHL-WT channels in the lipid bilayer throughout the scan. The positionof the lipid bilayer was recorded by imaging its position on an invertedmicroscope, and cyclodextrin binding events were observed electrically.Cyclodextrin binding frequency was determined by dividing the totalnumber of events by the number of aHL-WT channels in the lipid bilayer.FIG. 4C shows the diffusion limited localised binding of the twocyclodextrin analytes, plotting γCD and hβCD binding frequency in a scanbetween the two regions.

Bilayer Production Using a Pipette

With reference to FIG. 5, a hydrogel 11 is layered over a substrate 13to form a hydrated support which is then immersed in a lipid/oilsolution 7, such that a lipid monolayer forms on the upper surface ofthe hydrogel. An aqueous droplet 9 is partially ejected from a pipettetip 3 whilst the pipette tip 3 is immersed in a lipid/oil solution 7. Alipid monolayer forms on the surface of the droplet. The size of thedroplet is controlled by how much of the aqueous solution is pushed outof the pipette tip on the application of pressure in the pipette. Thesize of the area of contact between the droplet and hydrogel willdetermine the length of the bilayer formed. The area of contact can becontrolled by controlling the size of the droplet and the proximity ofthe pipette tip to the hydrogel.

The aqueous droplet 9 is contacted with the hydrogel 11 to form a lipidbilayer 1. A first electrode 5 in contact with the aqueous droplet 9 isused in conjunction with a second electrode (not shown) in contact withthe hydrogel 11 to measure electrical activity across the lipid bilayer1. The electrodes are Ag/AgCl.

By moving the pipette in the x/y plane the bilayer can be translocatedacross the surface of the hydrogel.

FIG. 6 uses inverted microscopy to visualise the formation of a lipidbilayer 801 between an aqueous droplet 809 and a transparent hydratedsupport (not shown) as the droplet and support are brought into contact.

Droplets in a Scanning ‘Pipette’

FIG. 7 illustrates that droplet-on-hydrated-support bilayers can beformed on a hydrated supported mounted in a translatable scanningpipette tip. More specifically, a hydrated support 111 is fixed to theopening of a pipette tip 103, and the pipette tip is filled with alipid/oil solution 107. An aqueous droplet 109 is immersed in thelipid/oil solution and contacted with the hydrated support 111 to form alipid bilayer 101. The lipid bilayer 101 can be used to scan or probe anaqueous system 113 by immersing the pipette tip 103 into an aqueoussystem 113. A pair of electrodes 105 is provided to measure electricalactivity across the lipid bilayer 101. One electrode 105 is in contactwith the aqueous droplet 109 and the other electrode 105 is in contactwith the aqueous system 113.

As long as the hydrated support is porous the bilayer can sensemolecules in the aqueous system which can pass though the pores. Thesize of the pores in the support can be controlled to filter whatmaterial actually reaches the bilayer.

Alternative Device for the Production of a Bilayer

FIG. 13A illustrates an alternative portable device for producing abilayer according to the invention. The device 801 is made of PMMA andcomprises a first screw 811, a second screw 812, a chamber 816 and ahole 820.

In use, the chamber 816 is filled with oil and a droplet or agarose ball822 is placed on the end of the second screw 812. The first screw 811 isadjustable to adjust the oil volume in the chamber 816. The second screw812 is adjustable to adjust the height of the droplet or agarose ball822.

To form a bilayer 830, the device 801, containing oil and including adroplet or agarose ball 822, is placed in a solution 825, the solutionmay be water. The first screw 811 is then raised to increase the volumein the chamber 816 which draws solution 825 into the chamber. When thesolution 825 contacts the droplet or agarose ball 822 a bilayer 830spontaneously forms. By adjusting the height of the second screw 812 thesize of the bilayer 830 can be adjusted. By connecting the second screw812 to an electrode, and placing a separate electrode in the solution825, electrical access to the bilayer 830 is allowed. Furthermore, whenthe device 801 is removed from the solution 825 the device holds ontoenough solution 825 to maintain the bilayer 830. This allows the bilayerto be removed from one solution and returned to a different solution.

FIG. 13B illustrates a bilayer 830 formed using the apparatus of FIG.13A. The edge 829 of the bilayer 830 is clearly visible.

Device for Producing an Array of Bilayers

FIG. 14A depicts a device which can be used to produce an array ofbilayers. The device comprises a base 842 and a lid 841. The base 840 isfilled with agarose in the lower channel, then filled with a lipid/oilsolution and left to equilibrate so that a monolayer forms on theagarose substrate. The lid 841 is dipped in a bulk aqueous solution, andthrough hydrophilic interaction with the plastic used to make the lid,small droplets remain on the lid. The lid is then lowered into the base,such that the droplets remain in oil for a time to equilibrate, beforethe lid is then lowered further to bring the droplets into contact withthe underlying agarose. An array of bilayers then forms spontaneouslywhere the droplets contact the agarose surface. By using electrodesconnected to each of the droplets through the lid, and with a commonelectrode in the agarose, each bilayer in the array is individuallyaccessible for electrical measurements. The nature of the device allowsindividual bilayers 844 to be imaged from below with a microscope, ascan be seen in FIG. 14B, in which an agarose wall 845, a plastic support846 and the suspended droplet 847 are also visible.

Device Capable of Perfusion of the Aqueous Phase

FIG. 15A illustrates how a basic device to producedroplet-on-hydrated-support bilayers according to the invention can beextended to provide control of the volume of the oil phase (in this casewith an adjustment screw) to manipulate the bulk liquid phase beingdrawn into the device.

In this embodiment a PMMA device 870 is attached to an underlyingagarose layer 872 mounted on glass 873. An internal cavity 875, channel,or network of channels is filled with a lipid/oil solution. The internaloil-filled cavity 875 is open to the environment at one end, andterminates with a mechanism to control the oil volume at the other end,in this case a screw. When the screw is wound in or unwound, the oil ispushed into or pulled out of the cavity. The means of adjusting volumeneed not be a screw, and can extend to any means of controlling thevolume of the oil, such as, it may be solid object such as a pin orneedle pushed into the sample, which could be actuated by a steppermotor or syringe driver, for example. Alternatively, the volume in thecavity may be controlled by a syringe upstream, or by a microfluidicpump. Alternatively, the cavity itself, or parts of the cavity, could bemade of a deformable material or include a membrane section, that whencompressed adjusts the volume of the oil. When the oil is pulled intothe cavity 875, bulk water or bulk aqueous volume 878 (added to theoutside of the device) is also pulled into the cavity. Where thewater/aqeuos volume contacts the underlying agarose a bilayer willspontaneously form.

With an electrode 879 in the bulk water or aqueous volume 878, andanother electrode 872 in the agarose 872 the bilayers are accessible forelectrical measurements. FIG. 15B illustrates an electrical traceproduced using the device of FIG. 15A showing an example of α-hemolysinchannels inserting into the bilayers (top), and binding cyclodextrins(bottom).

The bilayers can also be imaged using a microscope, see FIG. 15C, whichshow the bilayer 880 and the bilayer edge 882.

The bulk aqueous volume in the device can be a microfluidic channel 885as depicted in FIG. 16.

A device according to this embodiment, and illustrated in FIGS. 15A and16, has the advantage that the bulk aqueous volume is open to theexternal environment which means that there is easier access for addingcomponents to the system, which is difficult in a closed droplet system,and also that the volume can be perfused to fully exchange and alter thecontents of the aqueous volume.

Control on Bilayer Area and Capacitance Measurements

The droplet-on-hydrated-support bilayers subject of this application canbe very large, greater than 1 cm, or very small, less than 1 micrometre.The area of the bilayer can be adjusted very quickly by adjusting theheight of the droplet relative to the hydrated support. As isillustrated in FIG. 8, the size/area 215, 315, 415 of the lipid bilayer201 can be increased or decreased by moving the centre of the aqueousdroplet 109 towards or away from the hydrated support 111. In thisexample the droplet is moved by moving the electrode 205 in the droplettowards or away from the hydrated support 111.

By monitoring capacitance across the bilayer the area of the bilayer canbe monitored. Bilayers formed by the method of the invention shows alinear response of bilayer to area to capacitance. This relationship isregardless of whether the bilayer has been reformed and what areachanges the bilayer has been through.

The Production of Large Bilayers

By using devices such as those described herein, and the method of theinvention, larger bilayers, of 1 mm, 1 cm or more, can be formed. Forexample, by using the oil withdrawing techniques discussed herein, bulkwater can be drawn further and further down channels into the cavity ofa device to produce a large bilayer.

FIG. 17 shows a composite of images (the bilayer is too big to beobserved in its entirety) taken on a microscope using the 10× objectivein which the bilayer 900 is approximately 1 mm by 2 mm. The bilayer edge902 is visible as are small oil inclusions 903 and the bulk water inlet905.

FIG. 18 shows a slightly larger bilayer as the bilayer of FIG. 17 ispulled further into the channel, this bilayer is approximately 1 mm by 5mm in size. Bilayers of 1 mm by 1 cm in size were also produced, andlarger bilayers of many cms in size could be created. These largerbilayers may have a capacitance of ˜100,000 pFarads or more for aDiPhytanoylPC bilayer. This is in comparison to artificial bilayersproduced using previously known methods which are a few hundredmicrometers in diameter, and which have capacitances of a few hundredpFarads for DiphytanoylPC bilayers.

Bursting, Injection and Perfusion of Droplet-on-Hydrated-SupportBilayers

The droplet part of a droplet-on-hydrated-support bilayer can be burstwithout rupturing the bilayer. This is illustrated in FIG. 9 where thebursting of a cargo-carrying droplet into an existing droplet does notdisrupt the bilayer. More specifically, a second aqueous droplet 517carrying a cargo of reagents is shown bursting/fusing together with theexisting aqueous droplet 509 without rupturing the bilayer 501.

FIG. 10 illustrates that reagents can be introduced into an aqueousdroplet 609 by injection from a micro-pipette 619 or a micro-pipettewith a multi bore capillary 621. This offers a simple way of introducingcompounds into an existing droplet.

Droplet-on-Hydrated-Support Bilayers in Large Connected or UnconnectedNetworks

Multiple droplet-on-hydrated-support bilayers may be formed betweenindividual unconnected droplets on a hydrated support or betweenmultiple connected droplets on a hydrated support.

With reference to FIG. 11, multiple droplet-on-hydrated-support bilayers709 are dispersed on a single hydrated support 111. FIG. 11A shows aplurality of independent/separate aqueous droplets 709 on the hydratedsupport 111 all of which are forming a bilayer with the support.

FIG. 11B shows a plurality of aqueous droplets 709 connected in an arrayon the hydrated support 111, forming multiple bilayers between thedroplets 709 and with the hydrated support 111.

Such large arrays are suitable for fluorescent experiments. These arraysmay also be used for larger statistical studies. For example, an arrayof tiny droplets, from about 10s of nanolitres to 10s of picolitres, canbe made with the probability tuned that each contains only one moleculeof a given protein/reagent. Experiments can then be performed that trackthe turnover of an enzyme, for example, at the single molecule level.

Large arrays could also be used to fluorescently scan hydrogelscontaining isolated protein bands. For example if an array of dropletscontaining a Ca-sensitive fluorophore containing polymer is depositedover a gel containing calcium and isolated alpha-hemolysin channels inan electrophoretically focussed band, then droplets positioned over theprotein band would insert the aHL and become fluorescent as calciumentered the droplets.

Droplet-on-Hydrated-Support Bilayers for Fluorescence

Droplet-on-hydrated-support bilayers laid down on thin hydrated supportscan be fluorescently examined using total internal reflectionfluorescence (TIRF) microscopy.

FIG. 12, which illustrates total internal reflection fluorescencemicroscopy on a droplet-on-hydrated-support bilayer, a supportingsubstrate comprised of a thin layer of agarose is formed on a glasscoverslip. This thin substrate is rehydrated by filling a polymethylmethacrylate (PMMA) micro-channel device with aqueous agarose. Thedevice wells are filled with a solution of lipid in oil. An aqueousdroplet is placed on top of the hydrogel underneath the oil. A lipidbilayer forms at the interface between the two aqueous phases. Theevanescent field propagates into the droplet-on-hydrated-support bilayerilluminating the lipid bilayer and fluorophore-tagged biomolecules inthe droplet.

TIRF techniques may also be used in combination with other analysistechniques, for example, in combination with acquiring electrical data.The combination of data may provide improved information on proteinfunction.

Efficiency of Bilayer Formation

Experiments in which the hydrophilic body was a water droplet, and aplanar 1% agarose gel made with ultrapure water was the hydratedsupport, demonstrated that 20 water droplets all formed bilayers within1 minute of contact between the water droplet and the agarose gel. Thebilayers were observed to be intact after 2 weeks.

Concentration and Crystallisation of Membrane Proteins

FIG. 19 shows how bilayers according to the invention can be used toconcentrate membrane proteins. This could be a means to produce2-dimensional crystals or semi-ordered lattices of membrane proteins forfurther study. By inserting membrane proteins into a droplet-on-hydrogelbilayer, then shrinking the bilayer size by pulling the dropletperpendicularly from the surface, it is possible to drag membraneproteins inwards along the bilayer edge, without removing them from thebilayer. This results in a concentration of the inserted membraneproteins towards the centre of the bilayer.

FIG. 19 demonstrates the concentration of α-hemolysin pores embeddedinto a droplet-on-hydrogel bilayer. A 1% (w:v) thin agarose gel wasdeposited onto a coverslip and dehydrated, it was then rehydrated usinga 1.5% (w:v) agarose containing buffer and 750 mM CaCl₂. A dropletcontaining the calcium indicator dye Fluo-4 (25 μM), α-hemolysinheptamers, 1.5M KCl and a buffer, was used to create adroplet-on-hydrogel bilayer on this thin agarose gel.

Bilayer fluorescence (afforded by the non-chelating Fluo-4) was imagedas a circular disc through TIRF illumination. Upon insertion ofα-hemolysin pores into the bilayer, calcium flux into the droplet ispossible. In this example the flux was enhanced by the application of anexternally applied negative potential from electrodes inserted into thegel (ground) and droplet. This resulted in an enhanced fluorescenceemanating from the vicinity of the pores (imaged as a spot). This wasdue to the fluorescence of the Fluo-4 near the pore greatly increasingin intensity upon its immediate chelation of calcium upon entry into thedroplet.

FIG. 19 shows diffuse α-hemolysin pores (as fluorescent spots) 940diffusing in the bilayer using the method described above. The bilayeris then gradually shrunk 941, resulting in the pores being condensed asthey were dragged inwards by the encroaching bilayer edge 942. When thebilayer area was enlarged again 943, it was possible to see that thepores had been concentrated to where the bilayer edge was shrunk.Further concentration of the pores ensued 944 by repeating this process,leading to further condensation of the pores towards the centre of thebilayer area 945.

Discussion

Although droplet-on-hydrated-support bilayers represent a significantdeparture from conventional planar lipid bilayers, they are easier toprepare and work with, and are similarly amenable to single-channelrecording examinations of both major classes of membrane protein.Droplet-on-hydrated-support bilayers are more stable than planarbilayers, and in contrast to the typical planar bilayer lifetime of afew hours (Miller, C. 1986. Plenum Press: New York),droplet-on-hydrated-support bilayers are often still functional severalweeks after formation. This opens up avenues for long timescaleexperiments that have not been previously possible.

Droplet-on-hydrated-support bilayers also possess a number of otherunique properties: (i) the lipid bilayers can be moved across thesurface of a hydrated support. This has been exploited in hydrogelscanning experiments; (ii) droplet-on-hydrated-support bilayers providereliable access to stable bilayers over a much great size range (<1 μmto >1 mm) than possible using alternative techniques; (iii) the area ofthe lipid bilayer can be adjusted during an experiment. For example,this may be used to control the number of inserted proteins insingle-channel recording experiments; where the lipid bilayer isinitially enlarged to increase the probability of protein insertion,then rapidly reduced once a single protein has inserted to minimise thechances of further insertions. Reducing the lipid bilayer area may alsobe used to concentrate transmembrane proteins inserted in a lipidbilayer. This may provide an alternative means to crystallisemembrane-proteins, and for improving the probability of observingprotein-protein interactions; and (iv) the lipid bilayer can be removedand reformed many times without mixing the droplet and hydrogelsolutions. This may be used to reset single-channel recordingexperiments, as removing the lipid bilayer also appears to removeinserted transmembrane proteins. The fact that no contents mixing occursis also important for experiments where cross-contamination is an issue.

The sensitivity of droplet-on-hydrated-support bilayer gel scanningallows direct study of low levels of endogenous protein from cellextracts without the need for over-expression. In contrast, examiningproteins without over-expression using either traditional patchclamptechniques or planar lipid bilayers is difficult. Although whole-cellpatch clamping can examine low levels of endogenous protein, it is oftennecessary to compensate for other constituents of the system due to theheterogeneous nature of cell membranes (Hamill, O. P. et al., 1981.Pflugers Archly-European Journal of Physiology 391, 85-100; Ashley, R.H. 1995. IRL). It is possible to circumvent this problem usingartificial lipid bilayers, however, it is difficult to extract andconcentrate protein in sufficient quantities to successfullyreconstitute in these bilayers (Miller, C. 1986. Plenum Press: New York;Ashley, R. H. 1995. IRL).

Droplet-on-hydrated-support bilayer gel scanning may be incorporated inexisting proteomic methods that rely upon 2D gel electrophoresis toseparate complex mixtures of cellular components for the discovery andcharacterisation of new proteins (Palzkill, T. 2002 Proteomics, KluwerAcademic Publishers, Boston, London; Simpson, R. J. 2003. Proteins andproteomics: a laboratory manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). Droplet-on-hydrated-support bilayer gelscanning provides means to identify channel proteins in 2D gels. Theobservation that droplet-on-hydrated-support bilayer gel scanning doesnot appear to affect the proteins within the gel matrix means thatrepeated droplet-on-hydrated-support bilayer scanning of an individualgel under varying conditions, and subsequent analysis with conventionalproteomic techniques (e.g. mass spectrometry) can be performed.

This invention provides a new platform for high-throughput studies ofion-channels. In particular, the requirement for only nanolitre volumespermits the application of many established emulsion-based technologies(Joanicot, M. & Ajdari, A. 2005. Science 309, 887-888; Ahn, K. et al.,2006. Applied Physics Letters 88; Link, D. R. et al., 2006. AngewandteChemie-International Edition 45, 2556-2560; Hung, L. H. et al., 2006.Lab on a Chip 6, 174-178) for scaling-up and automatingdroplet-on-hydrated-support bilayers. For example, by combining flows oflipid/oil and water (Thorsen, T. et al., 2001. Physical Review Letters86, 4163-4166) thousands of droplets with a controlled size can becreated. Large numbers of droplet-on-hydrated-support bilayers may alsobe manipulated in an automated fashion with existing microfluidictechniques that can create and sort nanolitre droplets in oil (Ahn, K.et al., 2006. Applied Physics Letters 88; Link, D. R. et al., 2006.Angewandte Chemie-International Edition 45, 2556-2560).

The ability to image droplet-on-hydrated-support bilayers also allowsthe incorporation of fluorescence techniques. Single-channel recordingexperiments have provided a wealth of functional detail on manyion-channels, but it is difficult to link this to dynamic changes inprotein structure. Single-molecule fluorescence of labelled proteins isone possible method of providing additional structural and dynamicinformation. Moreover, droplet-on-hydrated-support bilayers allowssimultaneous optical and electrical measurements, which have thepotential to uncover new aspects of channel function that cannot beelucidated with the individual techniques alone (Borisenko, V. et al.,2003. Biophysical Journal 84, 612-622; Ide, T. & Yanagida, T. 1999.Biochemical and Biophysical Research Communications 265, 595-599; Ide,T. et al., 2002. Single Molecules 3, 33-42; Macdonald, A. G. & Wraight,P. C. 1995. Progress in Biophysics & Molecular Biology 63, 1-29; Suzuki,H. et al., 2007. Biosensors & Bioelectronics 22, 1111-1115; Suzuki, H.et al., 2006. Langmuir 22, 1937-1942); for example, the dynamics of thefolding and insertion of ion-channels.

Overall, the combination of enhanced stability, the ability tomanipulate the lipid bilayer, electrical access, and imaging demonstratethat droplet-on-hydrated-support bilayers according to the inventionprovide a versatile platform for examining many aspects of membraneprotein function.

1. A method for producing a bilayer of amphipathic molecules comprisingthe steps of: (i) providing a hydrated support in a hydrophobic medium,wherein a first monolayer of amphipathic molecules is present on thesurface of the hydrated support; (ii) providing a hydrophilic body in ahydrophobic medium, wherein a second monolayer of amphipathic moleculesis present on the surface of the hydrophilic body; and (iii) bringingthe first monolayer and the second monolayer into contact to form abilayer of amphipathic, wherein either: (a) the hydrophobic medium inwhich the hydrated support is provided contains amphipathic moleculesand the hydrophobic medium in which the hydrophilic body is providedcontains amphipathic molecules; or (b) the hydrated support containsamphipathic molecules and the hydrophilic body contains amphipathicmolecules. 2-7. (canceled)
 8. The method of claim 1 wherein theamphipathic molecules are lipid molecules.
 9. The method of claim 8wherein the lipid molecules are selected from the group comprising fattyacyls, glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenol lipids, saccharolipids, polyketides, phospholipids,glycolipids and cholesterol.
 10. The method of claim 8 wherein the lipidmolecules are selected from the group comprising monoolein;1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC);1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC);1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE);1-palmitoyl-2-oleoyl-phosphatidylethanolamine; and1-palmitoyl-2-oleoylphosphatidylglycerol (POPE/POPG) mixtures; ormixtures thereof.
 11. (canceled)
 12. The method of claim 1 wherein thehydrated support comprises a solid or a semi-solid substrate.
 13. Themethod of claim 1 wherein the hydrated support is hydrophilic. 14.(canceled)
 15. The method of claim 1 wherein the hydrated support isselected from the group comprising hydrogels, agarose, polyacrylamide,[cross-linked] polyethylene glycol, nitro-cellulose, polycarbonate,anodisc material, polyethersulphone, cellulose acetate, nylon, Naphionmaterials, mesoporous silica, water and glass.
 16. The method of claim 1wherein the hydrated support is a protein or analyte separation gel. 17.(canceled)
 18. The method of claim 1 wherein the hydrophilic bodycomprises a droplet of aqueous solution.
 19. The method of claim 18wherein the droplet is from 5 nm to 10 cm in diameter.
 20. The method ofclaim 1 wherein the hydrophilic body comprises a hydrated solid orsemi-solid support/substrate.
 21. The method of claim 1 wherein thehydrophobic medium is an oil.
 22. The method of claim 21 wherein the oilis a hydrocarbon.
 23. The method or bilayer of claim 21 wherein the oilis selected from the group comprising alkanes, alkenes, fluorinatedoils, silicone based oils and carbon tetrachloride.
 24. The method ofclaim 1 wherein the bilayer has a diameter from about 1 μm to greaterthan about 1 cm.
 25. (canceled)
 26. The method of claim 1 wherein amembrane-associated protein is present in at least one of the hydratedsupport, the hydrophilic body and the hydrophobic medium, saidmembrane-associated protein being capable of insertion into the bilayer.27. (canceled)
 28. The method of claim 26 wherein themembrane-associated protein is selected from the group comprising aselective or non-selective membrane transport protein, an ion channel, apore forming protein and a membrane-resident receptor.
 29. The method ofclaim 26 wherein the one or more protein is inserted into the bilayerafter the bilayer has formed.
 30. The method of claim 1 wherein the areaof the bilayer can be varied by varying the relative positions of thehydrophilic body and the hydrated substrate.
 31. The method of claim 1wherein the bilayer can be disassembled by removing contact between thehydrated support and the hydrophilic body.
 32. The method of claim 31wherein the bilayer can be reformed by restoring contact between a lipidmonolayer on the hydrated support and a lipid monolayer on thehydrophilic body. 33-56. (canceled)